Compositions and methods for administration of an enzyme to a subject&#39;s airway

ABSTRACT

Methods and composition for delivery of enzymes to a subject&#39;s airway. In some aspects, nebulized composition of enzymes, such as plasminogen activators are provided. In further aspects perfluorocarbon compositions comprising enzymes, such as plasminogen activators are provided. Compositions may, in some aspects, be used for the treatment of lung infections or acute lung injury, such as inhalational smoke induced acute lung injury (ISALI).

This application claims the benefit of U.S. Provisional PatentApplication No. 61/899,739, filed Nov. 4, 2013, the entirety of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology, drug delivery and medicine. More particularly, it concernscompositions and methods for the delivery of therapeutic enzymescompositions to a subjects respiratory system.

2. Description of Related Art

Many patients with severe burns and smoke exposure develop a severe formof ALI (Acute Lung Injury) called the Acute Respiratory DistressSyndrome (ARDS) that is associated with a mortality of 30-40%,protracted hospitalization and long-term morbidity. Inhalational smoke(IS)-induced ALI (ISALI) is characterized by severe airway obstruction,fibrinous airway casts or debris and alveolar fibrin deposition.Effective, specific treatment for ISALI is now lacking.

Burn injuries affect over 1 million patients in the United Satesannually and ISALI affects thousands of smoke-exposed patients incivilian and military practice annually (Enkhbaatar et al., 2004a).ISALI contributes to more than 3000 deaths and 17,000 fire-relatedinjuries in the United States annually and a fire-related mortality rateof 2-3/100,000 population, which is one of the highest in the developedworld [Committee on injury and poison prevention (2000) Pediatrics105:1355-1357]. Supportive care is suboptimal, protracted and expensive.Outcomes entail significant mortality and morbidity. Despite currentsupportive care including mechanical ventilation, the mortality rate ofARDS, including that associated with ISALI, approaches 30-40 percent(Phua et al., 2009).

These considerations demand the testing of new and potentially moreeffective therapy. ISALI is associated with severe respiratoryimpairment, protracted hospitalization and, often, the requirement formechanical ventilation. Long-term complications of ISALI includebronchial reactivity, accelerated pulmonary fibrosis and bronchiectasis.Among all forms of ALI, ISALI is especially prone to aberrant fibrinturnover including fibrin casts that form in the large airways andfibrin in the alveoli (Enkhbaatar et al., 2004a). Bronchial castsinterfere with gas exchange, often require bronchoscopic clearance andpromote atelectasis. While nebulized heparin and N-acetylcycteine areused in clinical practice, the efficacy of nebulized heparin in patientswith ISALI remains unproven in any randomized or prospectivelycontrolled clinical trials (Tuinman et al., 2012), nor has heparin alonebeen shown to improve ISALI when tested in our studies in sheep(Enkhbaatar et al., 2008a; Enkhbaatar et al., 2008b). Heparin does notclear established clots and nebulized heparin can initiate systemiccoagulopathy in ISALI (O'Donnell, 2012)

SUMMARY OF THE INVENTION

Provided herein is method of preparing an enzyme solution foradministration to a subject's airway that includes nebulizing the enzymesolution (e.g., using a vibrating mesh nebulizer). The enzyme can be atissue plasminogen activator, which includes a single chain urokinaseplasminogen activator (scuPA) and a tissue plasminogen activator (tPA).In some embodiments, the vibrating mesh nebulizer is an AERONEB®Professional Nebulizer or an EZ Breathe Atomizer.

Thus, in a first embodiment there is provided a method of preparing anenzyme solution for administration to a subject's airway comprisingnebulizing the enzyme solution to provide a nebulized solution. Incertain aspects, the enzyme may be a plasminogen activator, such as asingle chain urokinase plasminogen activator (scuPA) or a tissueplasminogen activator (tPA). In certain aspects, nebulizing the enzymesolution may be by using a vibrating mesh nebulizer. In some aspects,nebulizing the enzyme solution does not comprise use of a jet nebulizeror an ultrasonic nebulizer. In certain aspects, nebulizing an enzymesolution of the embodiments may comprise providing sufficientnebulization energy and/or time to provide a nebulized solution having amedian droplet size of between about 2.5 μm and 10 μm, 2.5 μm and 8 μm,or 3.0 μm and 6 μm. In some specific aspects, nebulizing the enzymesolution comprises obtaining a lyophilized enzyme composition,reconstituting the lyophilized enzyme composition in a solution (e.g.,an aqueous solution) to provide an enzyme solution, and nebulizing theenzyme solution. Thus, in a further embodiment, there is provided anebulized enzyme solution produced in accordance with the methods of theembodiments.

In still a further embodiment, there is provided a compositioncomprising a nebulized solution of scuPA or tPA. In some aspects,composition or enzyme solution of the embodiments may be an aqueoussolution. In certain aspects, the enzyme solution comprises aphysiologically acceptable salt concentration and/or a pH bufferingagent. For example, enzyme solution may be a sterile saline solution orphosphate buffered saline (PBS). In preferred aspects, the compositionor enzyme solution comprises scuPA.

In a further embodiment, a method treating a subject is providedcomprising administering a nebulized enzyme solution (e.g., a tPA and/orscuPA enzyme solution) to the airway of a subject in need thereof. Forexample, the subject may have an acute lung injury or infection. Instill further aspects, subject for treatment has inhalational smokeinduced acute lung injury (ISALI), chemical-induced lung injury, plasticbronchitis, severe asthma, or acute respiratory distress syndrome(ARDS). In this embodiment, the plasminogen activator is nebulized usingnebulizer, such as a vibrating mesh nebulizer (e.g., the AERONEB®Professional Nebulizer or the EZ Breathe Atomizer). The skilled artisanunderstands that any type of atomizer, such as a nebulizer, thatdelivers a therapeutically and pharmaceutically acceptable dose of theenzyme is suitable for use according to the embodiments.

In a further embodiment, there is provided a composition comprising aplasminogen activator and a perfluorocarbon (e.g., a “breathingliquid”). In some aspects, the plasminogen activator is scuPA and/ortPA. In some aspects, the perfluorocarbon may comprise a cycloalkylgroup. For example, the perfluorocarbon may be perfluorodecalin and/orperfluoro-octylbromide.

Another further embodiment of the invention provides a method fortreating a subject having a lung infection or lung injury comprisingadministering to the subject a therapeutically effective amount of acomposition comprising a plasminogen activator and a perfluorocarbon. Insome aspects, the plasminogen activator is a scuPA or a tPA. In certainaspects, the perfluorocarbon may be perfluorodecalin and/orperfluoro-octylbromide.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a graph showing that intratracheal delivery of recombinantscuPA in mice with bleomycin-induced ALI increases BAL uPA activity.

FIG. 2 shows that treatment of a sheep with nebulized scuPA provideddetectable uPA activity associated with human uPA antigen after scuPAtreatment (Lane 3). uPA antigen and activity were likewise found in lunghomogenates (Lane 4) from the scuPA-treated animal Lane 1: uPA standardand Lane 2: baseline uPA activity.

FIG. 3 is a schematic showing the methods of preparing several differentnebulized single chain urokinase plasminogen activator (scuPA)formulations.

FIG. 4 is a schematic showing the activity of the nebulized scuPAformulations prepared according to FIG. 3.

FIG. 5 is a schematic showing the activity of tissue plasminogenactivator (tPA) once mixed with perfluorodecalin (PFD) orperfluoro-octylbromide (PFB).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. THE PRESENT INVENTION

Provided herein is a method of preparing an enzyme solution foradministration to a subject's airway comprising nebulizing the enzymesolution using a nebulizer, such as a vibrating mesh nebulizer. It is asurprising finding of the present studies detailed herein thatnebulization of enzymes, such by using a vibrating mesh nebulizer,results in nebulized compositions that maintain significant enzymaticactivity levels. Also provided herein is a method of treating lunginjuries and infections, such as inhalational smoke induced acute lunginjury (ISALI) in a subject by administering to the subject atherapeutically effective amount of a nebulized plasminogen activatorvia an airway. In some cases, the plasminogen activator is nebulizedusing a vibrating mesh nebulizer. Further provided herein is acomposition comprising a plasminogen activator and a perfluorocarbon,and a method for using the plasminogen activator /perfluorocarboncomposition to treat lung injury and infection (e.g., ISALI). In someembodiments, an enzyme for use according to the embodiments is aproenzyme. In further aspects, the enzyme a plasminogen activator.

In still further embodiments, an enzyme for use herein is a plasminogenactivator selected from tPA and scuPA. The terms “tissue plasminogenactivator” and “tPA” are used interchangeably and refer herein to aserine protease (in some embodiments, EC 3.4.21.68) that can be involvedin the conversion of plasminogen to plasmin. It should be understoodthat the terms “tissue plasminogen activator” and “tPA” includerecombinant forms including, but not limited to, altepase, reteplase,tenecteplase, and desmoteplase. The terms “tissue plasminogen activator”and “tPA” further include the single chain form (sc-tPA), the two chainform (ds-tPA), and mixtures thereof In some embodiments, the tPA is ahuman tPA or a human-derived tPA. The terms “single chain urokinaseplasminogen activator” and “scuPA” are used interchangeably and referherein to a proenzyme of a urokinase serine protease (in someembodiments, EC 3.4.21.73), which serine protease can be involved in theconversion of plasminogen to plasmin. The “single chain urokinaseplasminogen activator” or “scuPA” can be activated by proteolyticcleavage between Lys158 and Ile159, resulting in two chains linked by adisulfide bond that form the serine protease enzyme. Example 3 and FIG.4 below describe the nebulization of scuPA using a vibrating meshnebulizer and the surprisingly high enzymatic activity achievedfollowing nebulization as compared to prior art methods of nebulization.In some embodiments, the vibrating mesh nebulizer is an AERONEB®Professional Nebulizer or an EZ Breathe Atomizer.

The term “enzyme solution” refers herein to any liquid formulationcontaining an enzyme that is suitable for nebulization. In someembodiments, the enzyme solution contains a pharmaceutically acceptablecarrier or excipient as defined herein. The enzyme solution isadministered to a subject's airway via inhalation or any other methodknown to those of skill in the art. The term “airway” refers herein toany portion of the respiratory tract including the upper respiratorytract, the respiratory airway, and the lungs. The upper respiratorytract includes the nose and nasal passages, mouth, and throat. Therespiratory airway includes the larynx, trachea, bronchi andbronchioles. The lungs include the respiratory bronchioles, alveolarducts, alveolar sacs and alveoli.

Also provided herein is a method of treating inhalational smoke inducedacute lung injury (ISALI) in a subject comprising administering to thesubject a therapeutically effective amount of a nebulized plasminogenactivator via an airway, wherein the plasminogen activator is nebulizedusing a vibrating mesh nebulizer. In some embodiments, the plasminogenactivator is selected from a tPA and a scuPA. In other or furtherembodiments, the vibrating mesh nebulizer is an AERONEB® ProfessionalNebulizer or an EZ Breathe Atomizer.

It should be understood that “treating ISALI” indicates partially orcompletely delaying, alleviating, mitigating or reducing the intensityof one or more attendant symptoms of a disorder or condition such as anISALI condition and/or alleviating, mitigating or impeding one or morecauses of a disorder or condition such as an ISALI condition. Treatmentsaccording to the invention may be applied preventively,prophylactically, pallatively or remedially. In some instances, theterms “treat,” “treating,” “treatment” and grammatical variationsthereof include partially or completely reducing a condition or symptomassociated with an ISALI condition as compared with prior to treatmentof the subject or as compared with the incidence of such condition orsymptom in a general or study population. In some embodiments, an ISALIcondition includes one or more of: reduced oxygenation, airwayobstruction (including a severe airway obstruction), fibrinous airwaycasts or debris, and alveolar fibrin deposition. Accordingly, treatingan ISALI condition includes one or more of improvement of oxygenation,reduced airway obstruction, reduced fibrinous airway casts or debris,and reduced alveolar fibrin deposition. In some embodiments, an ISALIcondition is treated with a reduced incidence of bleeding.

Further provided herein is a composition comprising a plasminogenactivator and a perfluorocarbon (PFC). In some embodiments, theplasminogen activator in the composition is selected from a tPA and ascuPA. In other or further embodiments, the PFC in the composition isselected from perfluorodecalin, perfluoro-1,3-dimethylcyclohexane,FC-75, perfluorooctane and perfluoro-octylbromide. In some aspects, PFCis or comprises a PFC having a cycloalkyl group, such asperfluorodecalin, perfluoro-1,3-dimethylcyclohexane or FC-75. It shouldbe understood that the plasminogen activator and PFC can be in any ratioor concentration. In some embodiments, the composition comprises aplasminogen activator at a concentration of approximately 0.005-0.040mg/mL of PFC.

Still further provided is a method of treating inhalational smokeinduced acute lung injury (ISALI) in a subject comprising administeringto the subject a therapeutically effective amount of a compositioncomprising a plasminogen activator and a PFC. Example 4 and FIG. 5demonstrate that a plasminogen activator, tPA, retained activity in aperfluorocarbon mixture. Further, the PFC and plasminogen activatoradditively foster airway debris removal as well as clearance of alveolarfibrin and improved outcome. Specifically, the PFC effectively deliversthe plasminogen activator which promotes 1) dissolution and dislodgementof the airway casts; and 2) removal of airway and alveolar debris whilesupporting respiratory gas exchange. Mechanistically, the PFCeffectively recruits lung volume. Given the low surface tension of thePFC liquid, the PFC distributes the plasminogen activator throughout thelung, potentially between casts and airway wall, thus breaking down thecasts as they are being formed while slowing formation of new casts. Asthe PFC volatizes from the lung, the plasminogen activator remains tofurther act to dissolve airway casts and alveolar fibrin. Upon redosingwith PFC suspensions, the PFC volumes not only deposit additional drugbut dislodge the casts and alveolar debris. Because the PFC isincompressible, it stents open damaged small airways and thereby aidsrecruitment. Although we are not bound by any certain mechanism, contactwith PFCs may also protect the underlying epithelium through attenuationof coagulation, which is initiated by tissue factor in the small airwaysand alveoli in virtually all forms of ALI. With in-line suctioning, thelower density debris float in the relatively more dense PFC,facilitating removal of airway fibrin cast fragments and debris.

Accordingly, in some embodiments of the method of administering aplasminogen activator and PFC composition, the plasminogen activator isselected from a tPA and a scuPA. In other or further embodiments of themethod of administering a plasminogen activator and PFC composition, thePFC in the composition is selected from perfluorodecalin andperfluoro-octylbromide.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims. Allpatents, patent applications, and publications referenced herein areincorporated by reference in their entirety for all purposes. Termdefinitions used in the specification and claims are as follows.

II. DEFINITIONS

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The term “administering” refers to an administration that is oral,topical, intravenous, subcutaneous, transcutaneous, transdermal,intramuscular, intra joint, parenteral, intra-arteriole, intradermal,intraventricular, intracranial, intraperitoneal, intralesional,intranasal, rectal, vaginal, by inhalation or via an implantedreservoir. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, and intracranial injections orinfusion techniques. In some embodiments, the administration is viainhalation of a nebulized composition.

The term “airway” refers herein to any portion of the respiratory tractincluding the upper respiratory tract, the respiratory airway, and thelungs. The upper respiratory tract includes the nose and nasal passages,mouth, and throat. The respiratory airway includes the larynx, trachea,bronchi and bronchioles. The lungs include the respiratory bronchioles,alveolar ducts, alveolar sacs and alveoli.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative.”“Mammal” for purposes of treatment refers to any animal classified as amammal, including a human, domestic and farm animals, nonhuman primates,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

The term “enzyme” refers herein to one or more polypeptides thatcatalyze a specific biochemical reaction or to a proenzyme. The term“proenzyme” refers to a biologically active substance that ismetabolized into an enzyme. In one embodiment, the enzyme is a tissueplasminogen activator (tPA). In other or further embodiments, the enzymeis a proenzyme and is a single chain urokinase plasminogen activator(scuPA).

The term “fibrinolysin” refers herein to any of several proteolyticenzymes that promote the dissolution of blood clots. A fibrinolysinincludes, but is not limited to, plasmin, tissue plasminogen activator(tPA, sc-tPA and dc-tPA), urokinase (uPA), and urokinase proenzymes(scuPA).

The term “identity” or “homology” shall be construed to mean thepercentage of amino acid residues in the candidate sequence that areidentical with the residue of a corresponding sequence to which it iscompared, after aligning the sequences and introducing gaps, ifnecessary to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.Sequence identity may be measured using sequence analysis software.

The terms “inhalational smoke induced acute lung injury” and “ISALI” areused interchangeably herein and refer to a form of acute lung injury(ALI) caused by smoke inhalation. ALI is also referred to as “mildARDS.” ALI can be defined by finding one or more of the followingconditions in a subject: 1) bilateral pulmonary infiltrates on chestx-ray, 2) when measured by right heart catheterization as clinicallyindicated, pulmonary capillary wedge pressure <18 mmHg (2.4 kPa), and 3)PaO2/FiO2 <300 mmHg (40 kPa). In some embodiments, treatment of ISALIincludes treatment of one or more of the following conditions: reducedoxygenation, airway obstruction (including a severe airway obstruction),fibrinous airway casts or debris, and alveolar fibrin deposition.

The terms “nebulizing,” “nebulized” and other grammatical variations,refer herein to the process of converting a liquid into small aerosoldroplets. In some embodiments, the aerosol droplets have a mediandiameter of approximately 2-10 μm. In some embodiments, the aerosoldroplets have a median diameter of approximately 2-4 μm.

The terms “perfluorocarbon” and “PFC” are used interchangeably and referherein to an organofluorine compound that contains predominantly carbonand fluorine. It should be understood that the term “perfluorocarbon” ismeant to include highly fluorinated molecules that contain molecules inaddition to carbon and fluorine, and are commonly referred to asfluorocarbons. Examples of perfluorocarbons include, but are not limitedto, perfluorodecalin, perfluoro-octylbromide, FC 77, PF 5060 and Rimar101. PFCs used according to the present invention share similarphysicochemical properties with respect to gas solubility, density andsurface tension but may differ with respect to radio-opacity andkinematic viscosity which could have an impact on visualization andmobility of airway casts during debridement. Each listed perfluorocarbonincludes all relevant isomers such as stereoisomers, enantiomers, anddiastereomers.

The term “plasminogen activator” refers to a serine protease polypeptidethat conversts plasminogen to plasmin, and includes, but is not limitedto, tPA, uPA (two chain or active forms) and a proenzyme scuPA asdefined herein.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” or“excipient” encompasses any of the standard pharmaceutical carriers,such as a phosphate buffered saline solution, saline (including sterilesaline), water, and emulsions, such as an oil/water or water/oilemulsion, where “oil” represents the water immiscible phase of theemulsion that is pharmaceutically acceptable, and various types ofwetting agents. The compositions also can include stabilizers andpreservatives. For examples of carriers, stabilizers and adjuvants, seeMartin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton(1975)).

The term “pharmaceutically acceptable salts” refers to any acid or baseaddition salt whose counter-ions are non-toxic to the subject to whichthey are administered in pharmaceutical doses of the salts. Specificexamples of pharmaceutically acceptable salts are known to those ofordinary skill in the art.

The terms “pharmaceutically effective amount,” “therapeuticallyeffective amount,” or “therapeutically effective dose” refer to theamount of a compound such as an ACPD composition that will elicit thebiological or medical response of a tissue, system, animal, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician.

The term “polypeptide” is used in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g. ester, ether,etc. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D or L optical isomers, and amino acid analogs and peptidomimetics.A peptide of three or more amino acids is commonly called anoligopeptide if the peptide chain is short. If the peptide chain islong, the peptide is commonly called a polypeptide or a protein.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” or “homology” to another sequencemeans that, when aligned, that percentage of bases (or amino acids) arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30,section 7.7.18, Table 7.7.1. Preferably, default parameters are used foralignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR.

The terms “prevent,” “preventing,” “prevention,” and grammaticalvariations thereof as used herein refer to a method of partially orcompletely delaying or precluding the onset or recurrence of a disorderor conditions and/or one or more of its attendant symptoms or barring asubject from acquiring or reacquiring a disorder or condition orreducing a subject's risk of acquiring or reacquiring a disorder orcondition or one or more of its attendant symptoms.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. Inpreferred embodiments, the subject is a human.

The terms “pharmaceutically effective amount,” “therapeuticallyeffective amount,” or “therapeutically effective dose” refer to theamount of a compound such as a tPA and/or scuPA composition that willelicit the biological or medical response of a tissue, system, animal,or human that is being sought by the researcher, veterinarian, medicaldoctor or other clinician.

The terms “single chain urokinase plasminogen activator” and “scuPA” areused interchangeably and refer herein to a proenzyme of a urokinaseserine protease polypeptide (in some embodiments, EC 3.4.21.73), whichserine protease can be involved in the conversion of plasminogen toplasmin, or to a proenzyme as described in U.S. Pat. No. 7,332,469,incorporated herein by reference. The “single chain urokinaseplasminogen activator” or “scuPA” can be activated by proteolyticcleavage between Lys158 and 11e159, resulting in two chains linked by adisulfide bond that form the serine protease enzyme. It should beunderstood that scuPA homologs are also included in the presentinvention. The term “scuPA homolog” refers herein to homologs,orthologs, and paralogs of the proenzyme of the urokinase serineprotease polypeptide identified as EC 3.4.21.73 and other sequenceshaving greater than 70% homology to the proenzyme of the urokinaseserine protease polypeptide identified as EC 3.4.21.73, or to aproenzyme as described in U.S. Pat. No. 7,332,469.

A “subject,” “individual” or “patient” is used interchangeably herein,which refers to a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets.

The term “therapeutically effective amount” includes that amount of acompound such as a tPA and/or scuPA composition that, when administered,is sufficient to prevent development of, or alleviate to some extent,one or more of the symptoms of an ISALI abnormality being treated. Thetherapeutically effective amount will vary depending on the compoundsuch as a tPA and/or scuPA composition, the disorder or conditions andtheir severity, the route of administration, the time of administration,the rate of excretion, the drug combination, the judgment of thetreating physician, the dosage form, and the age, weight, generalhealth, sex and/or diet of the subject to be treated.

The terms “tissue plasminogen activator” and “tPA” are usedinterchangeably and refer herein to a serine protease (in someembodiments, EC 3.4.21.68) that can be involved in the conversion ofplasminogen to plasmin. It should be understood that the terms “tissueplasminogen activator” and “tPA” include recombinant forms including,but not limited to, altepase, reteplase, tenecteplase, and desmoteplase.The terms “tissue plasminogen activator” and “tPA” further include thesingle chain form (sc-tPA), the two chain form (ds-tPA), and mixturesthereof In some embodiments, the tPA is a human tPA or a human-derivedtPA. It should also be understood that tPA homologs are also included inthe present invention. The term “tPA homolog” refers to homologs,orthologs, and paralogs of the tissue plasminogen activator polypeptideidentified as EC 3.4.21.68 and other sequences having greater than 70%homology to the tissue plasminogen activator polypeptide identified asEC 3.4.21.68. In some embodiments, the tPA is a single chain form suchas the ALTEPASE™ form.

The terms “treat,” “treating,” “treatment” and grammatical variationsthereof as used herein include partially or completely delaying,alleviating, mitigating or reducing the intensity of one or moreattendant symptoms of a disorder or condition such as an ISALI conditionand/or alleviating, mitigating or impeding one or more causes of adisorder or condition such as an ISALI condition. Treatments accordingto the invention may be applied preventively, prophylactically,pallatively or remedially. In some instances, the terms “treat,”“treating,” “treatment” and grammatical variations thereof includepartially or completely reducing a condition or symptom associated withan ISALI condition as compared with prior to treatment of the subject oras compared with the incidence of such condition or symptom in a generalor study population. In some embodiments, an ISALI condition includesone or more of: reduced oxygenation, airway obstruction (including asevere airway obstruction), fibrinous airway casts or debris, andalveolar fibrin deposition. In some embodiments, an ISALI condition istreated with a reduced incidence of bleeding.

The term “vibrating mesh nebulizer” refers herein to any nebulizer thatoperates on the general principle of using a vibrating mesh or platewith multiple aperatures (an aperture plate) to generate afine-particle, low-velocity aerosol. Some nebulizers may contain amesh/membrane with between 1000 and 7000 holes, which mesh/membranevibrates at the top of a liquid reservoir (see, e.g., U.S. Patent Publn.20090134235 and Waldrep and Dhand 2008, each incorporated herein byreference). In some embodiments, the vibrating mesh nebulizer is anAERONEB® Professional Nebulizer, Omron MICROAIR®, Pari EFLOW® or an EZBreathe Atomizer In some aspects, a vibrating mesh nebulizer has avibrating frequency of between about 50-250 kHz, 75-200 kHz 100-150 kHzor about 120 kHz. These devices have a high efficiency of deliveringaerosol to the lung and the volume of liquid remaining in these devicesis minimal, which is an advantage for expensive and potent compoundslike plasminogen activators.

III. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Intratracheal Delivery of Recombinant scuPA in Mice withBleomycin-Induced ALI Increases BAL uPA Activity

ALI was induced in C57/B6 mice with 2.5U/kg bleomycin at day 0 (n=10animals/group). Mice were treated daily with 25,000 U; 167 pg/mouse ofrecombinant human scuPA via a microsprayer (100 μl). At day 7, BAL wasobtained 4 hours after the last microsprayer administration of scuPA.uPA activity was measured using the amidolytic substrate S-2444(directly+after incubation with 1 mU/ml plasmin for 5 hours). Theresults are shown in FIG. 1. The increment of uPA activity after plasmintreatment suggested that some of the nebulized scuPA remained intactwithin the alveolar lining fluids and remained available for activationby plasmin in vivo. No pulmonary or systemic bleeding occurred. Thesedata confirm those of a previous report showing that nebulized scuPAincreases lavage fibrinolytic activity in trauma-induced AL and iswell-tolerated (Munster et al., 2002). The findings also show thatdurable fibrinolytic activity of scuPA (a substantive increase 4 hoursafter aerosolization) was generated in the lungs in ALI.

Example 2 Nebulized scuPA Lyses Airway Casts and is Detectable in BAL ofSheep with ISALI

A sheep was treated with nebulized scuPA (2 mg/treatment begun 4 hoursafter induction of ISALI and continued every 4 hours×48 hours. Airwaycast burden (obstruction score 12) fell into the range of sheep treatedwith nebulized tPA at 4 mg q 4 hours (vs. 20.7 in vehicle treated sheepwith ISALI) (Enkhbaatar et al., 2004b). As shown in FIG. 2, BAL(bronchoalveolar lavage) of sheep had no detectable baseline uPAactivity by fluorimetric analysis (Lane 2) but had detectable uPAactivity associated with human uPA antigen after scuPA treatment (Lane3) and uPA antigen and activity were likewise found in lung homogenates(Lane 4) from the scuPA-treated animal. Lane 1: uPA standard.

Example 3 Nebulized scuPA Lyses Airway Casts and is Detectable in BAL ofSheep with ISALI

Studies were also conducted on scuPA solutions containing 1 mg/mL ofscuPA dissolved in either physiological buffered saline or normalsaline, and then nebulized using two types of vibrating mesh nebulizers,the EZ Breathe Atomizer and the AeroNeb Pro nebulizer. scuPA readilydissolved in both liquid carriers. It was confirmed that the activity ofscuPA before and after nebulization was not affected by the nebulizingconditions (e.g., solution formation, shear and temperature from thenebulizing process in the nebulizer). Also, it was confirmed that themedian geometric particle size for the scuPA solutions was 3-4 micronswith a narrow and acceptable size distribution. The materials, methodsand results are provided below and a schematic of the procedure isprovided in FIG. 3.

Phosphate Buffered Saline and Sterile Saline Preparation:

The phosphate buffered saline (DPBS, Lot 14190-250, Gibco) was purchasedfrom Biostore at UT-Austin. The compositions of the PBS were as shown inTable 1.

TABLE 1 Molecular Concentration Components Weight (mg/L) mM InorganicSalts Potassium Chloride (KCl) 75 200 2.67 Potassium Phosphate 136 2001.47 monobasic (KH₂PO₄) Sodium Chloride (NaCl) 58 8,000 137.93 SodiumPhosphate dibasic 268 2,160 8.06 (Na₂HPO₄—7H₂O)

The pH of the PBS was 7.3±0.1. The sterile saline was purchased from B.Braun Medical Inc (Lot JIH573). Both preparations were stored at ambientroom temperature, and excessive heat was avoided.

Device Introductions:

The EZ Breathe Atomizer nebulizer and AeroNeb pro nebulizer were usedfor testing. The Aeroneb® Professional Nebulizer System (vibrating mesh,Aerogen, Galway) was a portable medical device for multiple patient use.The Aeroneb® is intended to aerosolize physician-prescribed medicationsfor inhalation that are approved for use with a general purposenebulizer. The EZ Breathe Atomizer (vibrating mesh, NephronPharmaceuticals Corporation, USA) is a device that is intended to sprayliquid medication in aerosol form into the air that a person willbreathe. These devices can be used by patients with and withoutmechanical ventilation, or other positive pressure breathing assistance.

Scu-PA Solutions Preparation:

Eight vials each containing 3.5 mg /mL of scu-PA were obtained andstored at −80° C. In order to make two kinds of scu-PA solutions, thesolutions were prepared in the following way:

A. scu-PA vials were held at room temperature (20° C.-25° C.) until theymelted into solution. Two mL of the solution was transferred from thevial to a new 10 mL vial using a 6 mL syringe.

B. Using a 6 mL syringe with attached 21-gauge needle, 3 mL of sterilephosphate buffered saline (or sterile saline) was injected into onevial. The contents were manually agitated until all scu-PA solution wasuniform. This solution was then diluted with sterile phosphate bufferedsaline (or sterile saline) to a final concentration of 1 mg/mL.

Geometric Particle-Size Distribution (PSD) Testing:

Both nebulizers were loaded with the two kinds of 5 mL scu-PA at aconcentration of 1 mg/mL as samples and pure saline and pure PBS asblank controls, separately (8 samples in total). The geometricparticle-size distribution (PSD) was determined using a MalvernSpraytec. A standard nebulization procedure was performed 5 times; eachtest lasted for 5 seconds. All determinations were carried out atambient room temperature, barometric pressure, and humidity.

Samples Collected from Nebulizer for Further Study:

After 10 seconds, the nebulized procedure was started, and run until theaerosol generation was stable, after which 500 μL samples of thenebulized output scu-PA (4 samples in total) were collected and thenfrozen in the −80° C. refrigerator. Six samples were prepared as shownin Table 2.

TABLE 2 With sterile phosphate buffered saline With sterile salineBefore nebulizing 1(1) 1(2) After nebulizing EZ Breathe Atomizer 1(3)1(5) nebulizer AeroNeb pro nebulizer 1(4) 1(6) Total: 6

Results:

Table 3 denotes geometric particle-size distribution (PSD) information(n=5) of the samples and FIG. 4 shows the activity of each samplefollowing nebulization.

TABLE 3 Nebulizer X(50%) X(10%) X(90%) name Sample Solvent μm μm μmAeroneb Pro PBS PBS 3.14 0.87 7.74 Saline Saline 3.25 0.92 8.66 scu-PAPBS 3.83 0.96 11.40 scu-PA Saline 3.60 0.91 11.48 EZ Breathe PBS PBS5.29 1.13 11.54 Atomizer Saline Saline 4.91 1.04 9.53 scu-PA PBS 4.791.16 11.17 scu-PA Saline 4.80 1.28 11.24

Based on these results, scuPA solutions are optimized for nebulizationfocusing on identifying the best nebulizer. Other parameters that arestudied for nebulizer administration include confirming the effect ofprocessing parameters (e.g., optimum solution composition for scuPAactivity, aerodynamic properties including fine particle fraction, massmedian aerodynamic diameter, and total emitted dose, temperature effectson scuPA activity during nebulization (e.g., using different nebulizermechanism types), and the effects of shear (e.g., atomization pressure,ultrasonic vibrations, mesh size) of the liquid on scuPA activity.

Example 4 Suspension of tPA in PFCs is stable and preserves tPA activity

FIG. 5 shows that suspension of tPA in PFCs is stable and that tPAactivity is preserved. For these studies, the tPA/PFC suspensions (18 mLof the tPA-PFC suspension made at 0.22 mg/mL) were injected through theendotracheal tube (7 mm adult endotracheal tube) during the 50 minutehold time at 37° C. using a 10 mL syringe and 21 gauge needle. It wasnoted that the tPA was adequately wetted and deagglomerated in the PFCs.Other parameters that are studied for tPA administration includeconfirming the effect of processing parameters (e.g., particle sizereduction of the tPA, viscosity of the PFC and resulting tPA-PFCsuspension, solids content of the tPA-PFC suspension and its effect onadministration during bronchoscopy on the tPA activity). The sameapproach is then used to analyze the formulations of the scuPA-PFCinterventions.

The PFC and fibrinolysins additively foster airway debris removal aswell as clearance of alveolar fibrin and improved outcome. Specifically,the PFC effectively delivers the fibrinolysins which promote 1)dissolution and dislodgement of the airway casts; and 2) removal ofairway and alveolar debris while supporting respiratory gas exchange.Mechanistically, the PFC effectively recruits lung volume. Given the lowsurface tension of the PFC liquid, the PFC distributes the fibrinolysinthroughout the lung, potentially between casts and airway wall, thusbreaking down the casts as they are being formed while slowing formationof new casts. As the PFC volatizes from the lung, the fibrinolysinremains to further act to dissolve airway casts and alveolar fibrin.Upon redosing with PFC suspensions, the PFC volumes not only depositadditional drug but dislodge the casts and alveolar debris. Because thePFC is incompressible, it stents open damaged small airways and therebyaids recruitment. Contact with PFCs may also protect the underlyingepithelium through attenuation of coagulation, which is initiated bytissue factor in the small airways and alveoli in virtually all forms ofALI. With in-line suctioning, the lower density debris float in therelatively more dense PFC, facilitating removal of airway fibrin castfragments and debris.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Committee on injury and poison prevention (2000) Pediatrics    105:1355-1357-   Enkhbaatar & Traber (2004) Clin. Sci. (Lond). 107:137-143-   Enkhbaatar et al. (2004) Shock 22:70-75-   Enkhbaatar et al. (2008). Clin. Sci. (Lond). 114:321-329-   Enkhbaatar, Herndon, and Traber (2008) J Burn Care Res. 30:159-162-   O'Donnell (2012). J. Pharm. Pract. 25:22-29-   Munster et al. (2002) Blood Coagul. Fibrinolysis 13:591-601-   Phua et al. (2009) Am J Respir Crit Care Med. 179:220-227-   Tuinman et al. (2012). Crit Care 16:R70-   Waldrep and Dhand, (2008) Current Drug Delivery, 5(2):114-119-   U.S. Patent Publn. 20090134235

What is claimed is:
 1. A method of preparing an enzyme solution foradministration to a subject's airway comprising nebulizing the enzymesolution to provide a nebulized solution.
 2. The method of claim 1,wherein the enzyme is a plasminogen activator.
 3. The method of claim 2,wherein the plasminogen activator is a single chain urokinaseplasminogen activator (scuPA) or a tissue plasminogen activator (tPA).4. The method of claim 2, wherein the plasminogen activator is a scuPA.5. The method of claim 1, wherein nebulizing the enzyme solution is byusing a vibrating mesh nebulizer.
 6. The method of claim 5, wherein thevibrating mesh nebulizer is an AERONEB® Professional Nebulizer or an EZBreathe Atomizer.
 7. The method of claim 5, wherein said nebulizing doesnot comprise use of a jet nebulizer or an ultrasonic nebulizer.
 8. Themethod of claim 1, wherein the enzyme solution is an aqueous solution.9. The method of claim 8, wherein the enzyme solution comprises aphysiologically acceptable salt concentration.
 10. The method of claim8, wherein the enzyme solution comprises a pH buffering agent.
 11. Themethod of claim 8, wherein the enzyme solution is phosphate bufferedsaline (PBS).
 12. The method of claim 8, wherein the enzyme solutioncomprises scuPA.
 13. The method of claim 1, wherein nebulizing theenzyme solution comprises: (i) obtaining a lyophilized enzymecomposition; (ii) reconstituting the lyophilized enzyme composition inan aqueous solution to provide an enzyme solution; and (iii) nebulizingthe enzyme solution.
 14. The method of claim 1, wherein nebulizing theenzyme solution comprises providing sufficient nebulization energyand/or time to provide a nebulized solution having a median droplet sizeof between about 2.5 μm and 10 μm.
 15. The method of claim 14, whereinnebulizing the enzyme solution comprises providing sufficientnebulization energy and/or time to provide a nebulized solution having amedian droplet size of between about 2.5 μm and 8 μm.
 16. The method ofclaim 15, wherein nebulizing the enzyme solution comprises providingsufficient nebulization energy and/or time to provide a nebulizedsolution having a median droplet size of between about 3.0 μm and 6 μm.17. A nebulized enzyme solution produced by a method according to anyone of claims 1 to
 16. 18. The method of any one of claims 1 to 16,further comprising administering the nebulized solution to the airway ofa subject in need thereof
 19. The method of claim 18, wherein thesubject has an acute lung injury or infection.
 20. The method of claim19, further comprising administering the nebulized solution to theairway of a subject having a chemical-induced lung injury.
 21. Themethod of claim 19, further comprising administering the nebulizedsolution to the airway of a subject having plastic bronchitis, asthma oracute respiratory distress syndrome (ARDS).
 22. The method of claim 19,further comprising administering the nebulized solution to a subjecthaving inhalational smoke induced acute lung injury (ISALI).
 23. Amethod of treating inhalational smoke induced acute lung injury (ISALI)in a subject comprising administering to the subject a therapeuticallyeffective amount of a nebulized plasminogen activator via an airway,wherein the plasminogen activator is nebulized using a vibrating meshnebulizer.
 24. The method of claim 23, wherein the plasminogen activatoris a single chain urokinase plasminogen activator (scuPA) or a tissueplasminogen activator (tPA).
 25. The method of claim 23, wherein theplasminogen activator is a scuPA.
 26. A composition comprising anebulized solution of single chain urokinase plasminogen activator(scuPA) or a tissue plasminogen activator (tPA).
 27. The composition ofclaim 26, wherein the solution is an aqueous solution.
 28. Thecomposition of claim 27, wherein the solution comprises aphysiologically acceptable salt concentration.
 29. The composition ofclaim 27, wherein the solution comprises a pH buffering agent.
 30. Thecomposition of claim 29, wherein the solution is phosphate bufferedsaline (PBS).
 31. The composition of claim 26, comprising scuPA.
 32. Thecomposition of claim 26, wherein the nebulized solution has a medianparticle size of between about 2.5 μm and 10 μm.
 33. The composition ofclaim 26, wherein the nebulized solution has a median particle size ofbetween about 2.5 μm and 8 μm.
 34. The composition of claim 26, whereinthe nebulized solution has a median particle size of between about 3.0μm and 6 μm.
 35. A composition for use in the treatment of acute lunginjury or lung infection, said composition comprising a nebulizedsolution in accordance with any one of claims 26-34.
 36. The compositionof claim 35, for use in the treatment of chemical-induced lung injury,plastic bronchitis, asthma, acute respiratory distress syndrome (ARDS)or inhalational smoke induced acute lung injury (ISALI).
 37. Acomposition for use in the treatment of inhalational smoke induced acutelung injury (ISALI), said composition comprising a nebulized solution inaccordance with any one of claims 26-34.
 38. A composition comprising aplasminogen activator and a perfluorocarbon.
 39. The composition ofclaim 38, wherein the plasminogen activator is a single chain urokinaseplasminogen activator (scuPA) or a tissue plasminogen activator (tPA).40. The composition of claim 38, wherein the plasminogen activator is ascuPA.
 41. The composition of claim 38, wherein the perfluorocarboncomprises a cycloalkyl group.
 42. The composition of claim 38, whereinthe perfluorocarbon is selected from perfluorodecalin andperfluoro-octylbromide.
 43. The composition of claim 38, wherein theperfluorocarbon comprises perfluorodecalin.
 44. A composition for use inthe treatment of acute lung injury or infection infection, saidcomposition comprising a composition in accordance with any one ofclaims 38-43.
 45. The composition of claim 44, for use in the treatmentof chemical-induced lung injury, plastic bronchitis, asthma, acuterespiratory distress syndrome (ARDS) or inhalational smoke induced acutelung injury (ISALI).
 46. A composition for use in the treatment ofinhalational smoke induced acute lung injury (ISALI), said compositioncomprising a composition in accordance with any one of claims 38-43. 47.A method for treating a subject having a lung infection or lung injury,comprising administering an effective amount of a composition inaccordance with any one of claims 38-43 to the airway of the subject.48. The method of claim 47, wherein the subject has chemical-inducedlung injury.
 49. The method of claim 48, wherein the subject has plasticbronchitis, acute respiratory distress syndrome (ARDS) or inhalationalsmoke induced acute lung injury (ISALI).
 50. A method for treating asubject having inhalational smoke induced acute lung injury (ISALI)comprising administering an effective amount of a composition inaccordance with any one of claims 38-43 to the airway of the subject.51. A method of treating inhalational smoke induced acute lung injury(ISALI) in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising aplasminogen activator and a perfluorocarbon.
 52. The method of claim 51,wherein the plasminogen activator is a single chain urokinaseplasminogen activator (scuPA) or a tissue plasminogen activator (tPA).53. The method of claim 51, wherein the plasminogen activator is ascuPA.
 54. The method of claim 51, wherein the perfluorocarbon isselected from perfluorodecalin and perfluoro-octylbromide.
 55. Themethod of claim 54, wherein the perfluorocarbon comprisesperfluorodecalin.